Use of alpha-linolenic acid metabolites for treatment or prevention of cancer

ABSTRACT

Cancer in a mammal can be treated or prevented by administering to a mammal in need thereof a cancer inhibiting amount of metabolite(s) of α-linolenic acid, such as stearidonic acid (18:4 n-3), eicosatetraenoic acid (20:4 n-3), docosapentaenoic acid (22:5 n-3) and mixtures thereof, especially metabolites including stearidonic acid.

FIELD OF THE INVENTION

[0001] This invention relates to the use of α-linolenic acid metabolitesfor the treatment of cancer. More specifically, this invention relatesto the use of stearidonic acid for treatment or prevention of cancer,particularly epithelial cell cancers such as, colon cancer, breastcancer, lung cancer and prostate cancer.

BACKGROUND OF THE INVENTION

[0002] There are two types of essential fatty acids (EFAs), the n-3 (orω-3) type derived from α-linolenic acid and the n-6 (or ω-6) typederived from linoleic acid. The starting polyunsaturated fatty acids(PUFAs) of these metabolic pathways (i.e., α-linolenic acid and linoleicacid) cannot be produced in the body, and therefore must be obtained inthe diet. The desaturation and elongation pathways for the n-3, n-6 andn-9 PUFAs are shown below.

[0003] An important factor providing evidence that dietary fats can havea significant effect on tumorigenesis is data which suggest that thetype of fat in the diet may be as important as the quantity of fat inmediating tumor promotion. In this regard, a great deal of attention hasbeen focused on PUFAs. Although the precise mechanisms responsible forthe effects of PUFAs are unknown, it has been suggested that PUFAeffects are mediated through arachidonic acid, possibly viaprostaglandins, HETEs and leukotrienes.

[0004] It has long been known that dietary n-3 PUFAs are very effectivein depressing tissue arachidonic acid content, and that the long chainn-3 PUFAs are more effective than α-linolenic acid. Whelan, J.,Broughton, K. S. and Kinsella, J. E., Lipids, Vol. 26, 119-126 (1991);Hwang, D. H., Boudreau, M. and Chanmugan, P., J. Nutr., Vol. 118,427-437 (1988). In addition, diets containing n-3 PUFAs, particularlythose found in fish oils (i.e., eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA)), are reported to diminish tumor formationand promotion, and n-3 PUFA intake is negatively correlated withchemically-induced tumorigenesis. Braden, L. M. and Carroll, K. K.,Lipids 21:285-288, 1986; Reddy, B., and Maruyama, H., Cancer Res.46:3367-3370, 1986; Minoura, T., Takata, T., Sakaguchi, M., Takada, H.,Yamamura, M., Hicki, K. and Yamamoto, J., Cancer Res. 48:4790-4794,1988; Nelson, R. L., Tanure, J. C., Andrianopoulos, G., Souza, G. andLands, W. E. M., Nutr. Cancer 11:215-220, 1988; Reddy, B. and Sugie, S.,Cancer Res. 48:6642-6647, 1988.

[0005] Tissue arachidonic acid content is correlated with eicosanoidbiosynthesis. Li, B. Y., Birdwell, C. and Whelan, J., J. Lipid. Res.,Vol. 35, 1869-1877 (1994). Eicosapentaenoic acid levels in colonicmucosal phospholipids are negatively associated with indices of cellproliferation. Lee, D. -Y. K., Lupton, J. R., Aukema, H. M. and Chapkin,R. S., J. Nutr., Vol. 123, 1808-1917 (1993). Conversely, arachidonicacid content in colonic mucosal phospholipids is associated with higherindices of cell proliferation. Lee, D. -Y. K., Lupton, J. R., Aukema, H.M. and Chapkin, R. S., J. Nutr., Vol. 123, 1808-1917 (1993).

[0006] More recently, Paulson et al. showed that a fish oil derivedconcentrate of eicosapentaenoic acid (EPA) and docosahexaenoic acid(DHA) decreased intestinal polyp formation and growth in Δ716 Apcknockout Min/+mice. Carcinogenesis, Vol. 18, 1905-1910 (1997).Similarly, Oshima et al. showed that dietary DHA-ethyl ester reducedintestinal polyp development in Δ716 Apc knockout Min/+mice.Carcinogenesis, Vol. 16, 2605-2607 (1995). Moser, A. R., Luongo, C.,Gould, K. A., McNeley, M. K., Shoemaker, A. R., Dove, W. F., Eur. J.Cancer, 31A(7-8), 1061-1064 (1995).

[0007] European patent application No. 0 440 307 A2 disclosescompositions for use in the treatment of breast cancer. The disclosedcompositions contain one or more metabolites of α-linolenic acid and oneor more metabolites of linoleic acid.

[0008] International Application No. 97/39749 describes methods for theprevention and treatment of cachexia and anorexia. Cachexia and anorexiaare said to be common conditions among cancer patients whose diseaseshave progressed to metastatic cancer. The disclosed methods involveadministering to an individual an oil blend containing n-6 and n-3 fattyacids, a source of amino-nitrogen which includes branched-chain aminoacids, and an antioxidant component.

[0009] U.S. Pat. No. 5,886,037 discloses food compositions for treatmentof various diseases which may be associated with the metabolic syndrome(syndrome X), including hyperlipoproteinaemia, obesity, hyperuricemia,hypertension, fatty liver, diabetes type II, insulin resistance andatherosclerotic vascular disease. The disclosed compositions containmedium-chain fatty acids and n-3 polyunsaturated long chain fatty acids.

[0010] U.S. Pat. No. 5,158,975 describes the use of stearidonic acid forprevention and treatment of inflammatory conditions, including allergicdisorders, skin disorders, rheumatic disorders, and those followingtrauma, shock and pathologies. Stearidonic acid (SDA) and itsmetabolites, EPA and DHA, are said to inhibit biosynthesis ofleukotrienes which are involved in the inflammation process.

[0011] U.S. Pat. No. 5,562,913 describes a method of treating n-6 or n-3essential fatty acid deficits in smokers. The method involvesadministering to the smoker a formulation containing an n-6 essentialfatty acid, an n-3 essential fatty acid, or a mixture of n-6 and n-3fatty acids.

SUMMARY OF THE INVENTION

[0012] The present invention is directed towards a method for treatingor preventing cancer in a mammal. The method involves administering to amammal in need thereof a cancer inhibiting amount of stearidonic acid(18:4 n-3), eicosatetraenoic acid (20:4 n-3), docosapentaenoic acid(22:5 n-3) or mixtures thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 illustrates the rate of uptake of radiolabeled ALA, SDA andEPA in HepG2 cells.

[0014]FIG. 2 illustrates the rate of metabolism of radiolabeled ALA tolong chain n-3 polyunsaturated fatty acid metabolites in HepG2 cells.

[0015]FIG. 3 illustrates the rate of metabolism of radiolabeled SDA tolong n-3 chain polyunsaturated fatty acid metabolites in HepG2 cells.

[0016]FIG. 4 illustrates the rate of metabolism of radiolabeled ALA andSDA to EPA in HepG2 cells.

[0017]FIG. 5 illustrates the rate of metabolism of radiolabeled ALA, SDAand EPA to DHA in HepG2 cells.

[0018]FIG. 6 illustrates the conversion of radiolabeled ALA, SDA and EPAto long chain n-3 polyunsaturated fatty acid metabolites in mouse liver.

[0019]FIG. 7 illustrates the conversion of radiolabeled ALA, SDA and EPAto long chain n-3 polyunsaturated fatty acid metabolites in mouse liver,corrected for recovery of radiolabeled fatty acid from liver tissue.

[0020]FIG. 8 illustrates the total amount of long chain n-3polyunsaturated fatty acids accumulated in mouse liver upon being fedthe US17 diet containing increasing amounts of ALA, SDA, EPA or DHA inthe ethyl ester form.

[0021]FIG. 9 illustrates the total amount of long chain n-3polyunsaturated fatty acids accumulated in rat liver upon being fed theUS17 diet containing increasing amounts of ALA, SDA, EPA or DHA in theethyl ester form.

[0022]FIG. 10 illustrates the effects of ALA, SDA, EPA and DHA fed asethyl esters on intestinal polyp number and size in the Min/+mouse modelof intestinal cancer.

[0023]FIG. 11 illustrates the effects of ALA, SDA, EPA and DHA fed asethyl esters on arachidonic acid content in the phospholipid (PL)fraction of mouse small intestine.

[0024]FIG. 12 illustrates the effect of SDA fed as an ethyl ester onprimary tumor growth in the nude mouse/HT-29 cancer model.

DETAILED DESCRIPTION OF THE INVENTION

[0025] As used herein, the term “treatment” includes partial or totalinhibition of growth, spreading or metastasis of benign tumors,cancerous tumors and polyps, as well as partial or total destruction oftumor and polyp cells. The term “prevention” includes either preventingthe onset of clinically evident tumors or polyps altogether orpreventing the onset of a preclinically evident stage of tumor or polypdevelopment in individuals at risk. The term “prevention” also includesprevention of initiation for malignant cells or to arrest or reverse theprogression of premalignant cells to malignant cells. This includesthose at risk for developing tumors and/or polyps.

[0026] The present inventors have discovered that administration ofstearidonic acid (SDA; 18:4 n-3) to mammals raises the tissue levels ofeicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in themammals to a higher level than does administration of an equivalentamount of α-linolenic acid (ALA; 18:3 n-3). In addition, the presentinventors have discovered that SDA is converted to EPA and DHA at a rateand efficiency that permits the use of SDA as a precursor to EPA and DHAfor treating or preventing cancer. The present inventors also havesurprisingly discovered that dietary SDA may be more effective than evendietary EPA and DHA in inhibiting tumorigenesis of the large intestine.

[0027] Moreover, administering SDA to a patient rather than EPA and/orDHA takes advantage of “physiological channeling”, in which themetabolism of SDA to EPA and DHA is ultimately controlled by the body'sfatty acid metabolism, leading to optimal distribution in lipid poolsaffecting tumorigenesis (e.g., competing with arachidonic acidmetabolism). This metabolic control may result in a more efficaciousdistribution of EPA and DHA than that provided by direct administrationof EPA and/or DHA. Additionally, because SDA has a smaller chain length,and fewer unsaturated bonds than EPA and DHA, SDA may exhibit moredesirable organoleptic properties than either EPA or DHA. Consequently,SDA may serve as an especially attractive substitute for EPA and DHA ina number of applications, including for example, functional foods ornutritional supplements.

[0028] Cancers which may be treated or prevented by the method of thisinvention include epithelial cell cancers, such as colon cancer, breastcancer, prostate cancer and lung cancer. Other cancers which may betreated or prevented by the method of this invention include braincancer, bone cancer, adenocarcinoma, gastrointestinal cancers such aslip cancer, mouth cancer, esophageal cancer, small bowel cancer andstomach cancer, liver cancer, bladder cancer, pancreatic cancer, ovariancancer, cervical cancer, renal cell carcinoma, and skin cancer such assquamous cell and basal cell cancers.

[0029] When administered to a mammal, SDA may be in any biologicallyactive form. For example, SDA may be in the carboxylic acid form, or mayinstead be in the form of a lipid, a carboxylate salt, an ester, anamide or some other pharmacologically acceptable carboxylic acidderivative. Besides SDA, other metabolites of ALA which may be used inthe present invention as precursors to EPA and DHA to treat or preventcancer include eicosatetraenoic acid (20:4n-3), docosapentaenoic acid(DPA n-3; 22:5n-3) and mixtures thereof.

[0030] The α-linolenic acid metabolites may be administered in the formof a pharmaceutical, nutritional or food preparation. Those of ordinaryskill in the art of preparing pharmaceutical formulations can readilyformulate pharmaceutical compositions having one or more metabolites ofα-linolenic acid using known excipients (e.g., saline, glucose, starch,etc.). The pharmaceutical compositions may be formulated according tothe desired method of administration. For example, pharmaceuticalformulations containing one or more α-linolenic acid metabolites may beprepared for oral, enteral, parenteral or rectal administration.

[0031] Similarly, those of ordinary skill in the art of preparingnutritional formulations (e.g., nutritional supplements) can readilyformulate nutritional compositions having α-linolenic acid metabolites.And those of ordinary skill in the art of preparing food or foodingredient formulations can readily formulate food compositions or foodingredient compositions having α-linolenic acid metabolites.

[0032] The dosing regimen will depend upon the particular α-linolenicacid metabolite administered and the desired therapeutic or prophylacticeffect. Typically, the amount of α-linolenic acid metaboliteadministered will be between about 1 mg/Kg/day and about 300 mg/Kg/day.Preferably, the amount of the metabolite administered is between about10 mg/Kg/day and about 150 mg/Kg/day. The desired dosage may beadministered as most efficacious, generally from 1-5 doses per day,desirably from 1-3 doses per day.

[0033] Preferably, the α-linolenic acid metabolite administered to themammal is SDA or a combination of SDA and at least one other ALAmetabolite.

[0034] The examples which follow are intended to illustrate certainpreferred embodiments of the invention, and no limitation of theinvention is implied. The n-3 PUFAs used in all of the followingexamples were in the free acid form (100% pure) when used in cellculture and in the ethyl ester form (>85% pure) when administered invivo. The ethyl esters of stearidonic acid (SDA-EE), eicosapentaenoicacid (EPA-EE) and docosahexaenoic acid (DHA-EE) were derived from fishoil, and were obtained from KD Pharma (Bexbach, Germany). The ethylester of stearidonic acid was further purified by Callanish, Ltd.(Scotland, U.K.) to increase the SDA-EE content from approximately 60%to 85% and also to decrease the EPA-EE content from approximately 8% to0.2%. The ethyl esters of α-linolenic acid (ALA-EE) and γ-linolenic acid(GLA-EE), which were derived from plant oils, were at least 95% pure andwere purchased from Callanish, Ltd. Administration of the fatty acidethyl esters in rodents was scaled allometrically by caloric equivalencyto reflect the human equivalent amount of fatty acid consumed per day(=g/day human equivalent dose).

EXAMPLE 1 The US17 Diet

[0035] In order to study the effects of PUFAs on colon tumor formationand promotion in rodents, a diet (the “US17 diet”) was designed to mimicthe human western diet. The human western diet contains high levels ofsaturated fatty acids and linoleic acid, both of which have been linkedto cancer formation. The components of the US17 diet are set forth inTables 1-6, below. TABLE 1 Ingredients of the US17 diet. IngredientAmount (grams) Casein, Alcx 200 L-Cystine 3 Corn Starch 240 Maltodextrin10 75 Sucrose 100 Cellulose 50 Cocoa Butter (Deodorized) 37.5 LinseedOil 4.5 Palm Oil (Bleached, deodorized) 52.5 Safflower Oil, USP 28.5Sunflower Oil, Trisun Extra 27 t-BHQ 0.03 Salts (See Table 2) 10Dicalcium Phosphate 13 Calcium carbonate 5.5 Potassium citrate(Monohydrate) 16.5 Vitamins (See Table 3) 10 Choline bitartrate 2α-Vitamin E acetate (500 IU/gm) 0.13 Total (grams) 875.16

[0036] TABLE 2 Salt mixture of the US17 diet Ingredient Amount (gm)Sodium Chloride 25.90 Magnesium Oxide 4.19 Magnesium Sulfate.7H₂O 25.76Chromium Potassium Sulfate.12H₂O 0.19 Cupric Carbonate 0.10 SodiumFluoride 0.02 Potassium Iodate 0.003 Ferric Citrate 2.10 ManganousCarbonate 1.23 Ammonium Molybdate .4H₂O 0.03 Sodium Selenite 0.003 ZincCarbonate 0.56 Sucrose 39.91 Total 100

[0037] TABLE 3 Vitamin mixture of the US17 diet Ingredient Amount (gm)Vitamin A Palmitate 0.08 500,000 IU/gm Vitamin D3 0.10 100,000 IU/gmVitamin E Acetate 1.00 500 IU/gm Menadione Sodium Bisulfite 0.008 Biotin1.0% 0.20 Cyanocobalamin 0.1% 0.10 Folic Acid 0.02 Nicotinic Acid 0.30Calcium Pantothenate 0.16 Pyridoxine-HCl 0.07 Riboflavin 0.06 ThiaminHCl 0.06 Sucrose 97.84 Total 100

[0038] TABLE 4 Fatty acid content of the US17 diet Fatty Acid AmountC14, Myristic  0.7 gms C16, Palmitic 34.6 gms C16:1, Palmitoleic  0.2gms C18:2, Stearic 17.5 gms C18:1, Oleic 60.5 gms C18:2, Linoleic 30.2gms C18:3, Linolenic  3.1 gms C20, Arachidic  0.4 gms Saturated 36.1weight % Monounsaturated 41.3 weight % Polyunsaturated 22.6 weight %

[0039] TABLE 5 Nutritional content of the US17 diet Nutrient AmountProtein 22.7 weight % Carbohydrate 48.6 weight % Fat 17.1 weight % Fiber5.7 weight % Protein 20.7 kcal % Carbohydrate 44.2 kcal % Fat 35.1 kcal%

[0040] TABLE 6 Comparison between rodent US17 diet and human, westerndiet Kcal % Rodent diet Nutrient (US17) Human Diet Protein 21 15Carbohydrate 44 50 Fat 35 35 Fatty Acid Composition <C16 0.2 1.6 16:08.6 7.9 18:0 4.3 3.9 18:1 n-9 (oleic acid 14.6 14.0 cassette) 18:2 n-67.0 6.9 18:3 n-3 0.7 0.7 n-6:n-3 ratio 10:1 10:1

[0041] The fatty acid test agent was substituted for oleic acid (=oleicacid cassette) and the dose, when scaled allometrically, was in therange readily consumed by humans (i.e., 0.1 to 10 g/day).

EXAMPLE 2 Uptake of ¹⁴C-ALA, ¹⁴C-SDA and ¹⁴C-EPA by HepG2 Cells

[0042] The uptake of stearidonic acid by HepG2 cells was compared tothat of α-linolenic acid and eicosapentaenoic acid.

[0043] To a culture medium containing HepG2 cells was added 20 μM¹⁴C-ALA, ¹⁴C-SDA or ¹⁴C-EPA complexed to fatty acid free BSA. The amountof ¹⁴C-ALA, ¹⁴C-SDA or ¹⁴C-EPA taken up by the HepG2 cells was measured6 hours, 24 hours and 48 hours after addition of the fatty acid. Ascintillation counter was used to measure the total amount ofradioactivity in the HepG2 cells and the amount remaining in the medium.

[0044] The results of these measurements are shown in FIG. 1. As can beseen in FIG. 1, ¹⁴C-ALA, ¹⁴C-SDA and ¹⁴C-EPA were taken up equally byHepG2 cells. Approximately 95% of each radiolabeled fatty acid was takenup by the cells within the first six hours of incubation.

EXAMPLE 3 Metabolism of Stearidonic Acid to Long Chain n-3Polyunsaturated Fatty Acids in HepG2 Cells

[0045] The metabolism of stearidonic acid to long chain n-3 PUFAs(eicosatetraenoic acid (20:4n-3), eicosapentaenoic acid (20:5n-3),docosapentaenoic acid (22:5n-3), and docosahexaenoic acid (22:6n-3)) inHepG2 cells was compared to that of α-linolenic acid.

[0046] HepG2 cells were allowed to take up ¹⁴C-ALA or ¹⁴C-SDA asdescribed in Example 2. The total amount of ¹⁴C-EPA, ¹⁴C-DPA and ¹⁴C-DHApresent in the HepG2 cells was measured 6 hours, 24 hours and 48 hoursafter addition of the fatty acid by argentation thin layerchromatography (TLC). The amount of each fatty acid present as a band onthe TLC plate was quantified by electronic autoradiography using anInstant Imager supplied by Packard (Meriden, Conn.).

[0047] The results of these measurements are shown in FIGS. 2 and 3.FIG. 2 shows the metabolism of ALA to long chain n-3 PUFAs. FIG. 3 showsthe metabolism of SDA to long chain n-3 PUFAs. A comparison of FIG. 2 toFIG. 3 shows that the metabolism of SDA in Hep2G cells to long chain n-3PUFAs is faster than that of ALA. Nearly 95% of the ¹⁴C-SDA wasmetabolized to ¹⁴C-fatty acid end products (i.e., EPA or DHA) or ¹⁴Cfatty acid intermediates (i.e., 20:4 n-3, 22:5 n-3 and 24:5 n-3).¹⁴C-SDA was metabolized more efficiently to ¹⁴C-EPA than was ¹⁴C-ALA(55% versus 24%).

EXAMPLE 4 Metabolism of Stearidonic Acid to EPA and DHA in HepG2 Cells

[0048] The metabolism of stearidonic acid to eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6 n-3)) in HepG2 cells was compared tothat of α-linolenic acid.

[0049] HepG2 cells were allowed to take up ¹⁴C-ALA, ¹⁴C-SDA or ¹⁴C-EPAas described in Example 2. The amount of ¹⁴C-EPA and ¹⁴C-DHA present inthe HepG2 cells was measured 6 hours, 24 hours and 48 hours afteraddition of the fatty acids by argentation thin layer chromatography(TLC) as described in Example 3. The amount of ¹⁴C-EPA and ¹⁴C-DHApresent as bands on the TLC plate were quantified by electronicautoradiography using an Instant Imager as described in Example 3.

[0050] The results of these measurements are shown in FIGS. 4 and 5.FIG. 4 shows the metabolism of radiolabeled ALA and SDA to EPA.Radiolabeled EPA was included as a control to evaluate its maintenancein HepG2 cells over time. FIG. 5 shows the metabolism of radiolabeledALA, SDA and EPA to DHA. FIG. 4 shows that SDA was metabolized moreefficiently to EPA than was ALA (55% versus 24%). The amount of EPAderived from SDA was actually quite similar to the amount of EPA thatremained after incubation with EPA itself (55% versus 63%). FIG. 5 showsthat SDA was metabolized to DHA more efficiently than was ALA (6% versus3%). In comparison, approximately 11% of EPA was metabolized to DHA.Overall, the results showed that SDA was metabolized to EPA and furtherto DHA at a rate that was approximately twice that of ALA.

EXAMPLE 5 Metabolism of Stearidonic Acid in Mice (Time Course AnalysisUsing Radiolabeled Fatty Acids)

[0051] The metabolism of stearidonic acid to long chain n-3polyunsaturated fatty acids (i.e., eicosapentaenoic acid (EPA),docosapentaenoic acid (DPA n-3), and docosahexaenoic acid (DHA)) inmouse liver was compared to that of α-linolenic acid, eicosapentaenoicacid and docosahexaenoic acid. The method used was a time course studyusing ¹⁴C-labeled fatty acids.

[0052] Mice were fed the US17 diet for a period of one month in order toachieve steady-state fatty acid metabolism. After achieving steady-statefatty acid metabolism, the mice were administered an intraperitonealinjection containing 10 μCi of ¹⁴C-ALA, ¹⁴C-SDA, ¹⁴C-EPA or ¹⁴C-DHA.Mice were sacrificed 3 hours, 8 hours or 24 hours post-injection, andthe total amount of ¹⁴C-EPA, ¹⁴C-DPA n-3 and ¹⁴C-DHA was measured byargentation thin layer chromatography followed by direct electronicautoradiography, as described in Example 3. The results of theseexperiments are shown in FIG. 6. FIG. 6 shows the order of metabolism ofthe PUFAs to be DHA=EPA>SDA>ALA at 24 hours. This difference inmetabolism rates was especially magnified when the conversion toEPA+DPA+DHA was adjusted to ¹⁴C counts recovered from liver tissue. WithALA, the recovery was notably lower, most probably due to the propensityof ALA to undergo beta-oxidation. This adjustment is shown in FIG. 7,which further shows that SDA is metabolized to long chain n-3 PUFAs moreefficiently than ALA in vivo.

EXAMPLE 6 Metabolism of Stearidonic Acid in Mice (End Point AnalysisUsing Cold Fatty Acid Ethyl Esters)

[0053] The metabolism of n-6 and n-3 PUFAs in rats and mice is similarto that of humans. Lands, W. E. M., Morris, A., Libelt, B., Lipids, Vol.25(9), pp. 505-516 (1990). As such, fatty acid metabolic results fromstudies with rats and mice would be predicted to be similar in humans.

[0054] The metabolism of stearidonic acid to long chain n-3polyunsaturated fatty acids (i.e., eicosapentaenoic acid (EPA),docosapentaenoic acid (DPA n-3) and docosahexaenoic acid (DHA)) in mouseliver was compared to that of α-linolenic acid, eicosapentaenoic acidand docosahexaenoic acid. The method used was a non-radioactive, doseresponse, metabolic end-point study.

[0055] Mice were fed a US17 diet containing α-linolenic acid ethyl ester(ALA-EE), stearidonic acid ethyl ester (SDA-EE), eicosapentaenoic acidethyl ester (EPA-EE), or docosahexaenoic acid ethyl ester (DHA-EE) in anamount equivalent to a human western diet containing 1, 3 or 10 g/day ofthe fatty acid (g/day human equivalent dose). In order to maintain a 17%fat (37 en %) content in the US17 diet, oleic acid (18:1 n-9), as anoleic acid cassette, was removed from the US17 diet in an amount equalto the amount of fatty acid ester that was added. Oleic acid wasselected as the replacement fatty acid because literature reportsindicate that oleic acid is neutral with respect to inflammation andcancer.

[0056] After one month on the respective US17-based diets, the mice weresacrificed and the fatty acid composition of each of their livers wasanalyzed by gas chromatography. The results of these analyses arepresented in FIG. 8.

[0057]FIG. 8 shows that the sum of the long chain n-3 PUFAs (i.e.,EPA+DPA n-3+DHA) increased dose dependently in liver tissue in thefollowing rank order: DHA-EE>EPA-EE>SDA-EE>ALA-EE. These results showedthat SDA was metabolized to long chain n-3 polyunsaturated fatty acidsbetter than ALA. FIG. 8 also shows that each of the dietary n-3 PUFAsdecreased the level of arachidonic acid in liver tissue dosedependently. This is significant because arachidonic acid metabolites(e.g., prostaglandins, leukotrienes, and HETEs (hydroxyeicosatetraenoicacid)) are correlated with tumorigenesis. The group “basal” refers tomice that were fed the standard rodent chow diet just prior to switchingto diets that were US17 based. The results showed that the level of thesum of the long chain n-3 PUFAs or arachidonic acid was the same,indicating that the US17 diet did not significantly alter fatty acidcomposition compared to the standard rodent chow diet.

EXAMPLE 7 Metabolism of Stearidonic Acid in Rats (End Point AnalysisUsing Cold Fatty Acid Ethyl Esters)

[0058] The metabolism of stearidonic acid to long chain n-3polyunsaturated fatty acids (i.e., eicosapentaenoic acid (EPA),docosapentaenoic acid (DPA n-3) and docosahexaenoic acid (DHA)) in ratliver was compared to that of α-linolenic acid, eicosapentaenoic acidand docosahexaenoic acid. The method used was as described in Example 6except that rats were used in place of mice. The results of theseanalyses are presented in FIG. 9.

[0059]FIG. 9 shows that the sum of the long chain n-3 PUFAs (i.e.,EPA+DPA n-3, and DHA) accumulated dose dependently in liver tissue inthe following rank order: DHA-EE>EPA-EE>SDA-EE>ALA-EE. These resultsshowed that SDA was metabolized to long chain n-3 polyunsaturated fattyacids better than ALA. FIG. 9 also shows that each of the dietary n-3PUFAs decreased the level of arachidonic acid in liver tissuedose-dependently. SDA caused a greater decrease in the level ofarachidonic acid than did either ALA or EPA.

EXAMPLE 8 Effect of n-3 and n-6 PUFAs on Intestinal Cancer in theMin/+Mouse Model

[0060] The efficacy of select n-3 and n-6 polyunsaturated fatty acidswas evaluated in the Min/+mouse model of intestinal cancer. Thefollowing fatty acid ethyl esters were tested for their effect onintestinal polyp formation: 1) ALA-EE; 2) SDA-EE; 3) EPA-EE; 4) DHA-EE;5) GLA-EE (α-linolenic adid, 18:3 n-6); and 6) CLA-EE (conjugatedlinoleic acid; c9t11-18:2 (77%) +c9c11-18:2 (18%)+other isomers (5%)).

[0061] These fatty acid ethyl esters were added to the US17 diet toprovide 10 g/day human equivalent dose (3% wt.). The NSAID, sulindac(320 ppm), served as the positive control. In order to maintain a 17%fat (37 en %) content in the US17 diet, oleic acid (18:1 n-9) wasremoved from the US17 diet in an amount equal to the amount of the fattyacid ethyl ester that was added. Mice were received at approximatelyfive weeks of age and were fed the test diets upon receipt. After sevenweeks on the respective test diet, the mice were sacrificed and theintestinal polyps were counted and measured. The results of theseanalyses are presented in Table 6. TABLE 6 Effect of various fatty acidson tumor size and number in the large and small intestine of Min/+ miceUS17 ALA CLA DHA EPA GLA SDA Sulindac (n = 10) (n = 10) (n = 9) (n = 10)(n = 10) (n = 9) (n = 10) (n = 9) Total large   13(7/10)    8(5/10)  10(5/9)   18(9/10)    9(6/10)   11(6/9)    2(2/10)   2(2/9) intestinetumors/group Avg. large  1.3 ± 0.6^(ab)  0.8 ± 0.3^(bcd)  1.1 ±0.4^(abcd)  1.8 ± 0.4^(a)  0.9 ± 0.3^(abcd)  1.2 ± 0.4^(abc)  0.2 ±0.1^(d)  0.2 ± 0.2^(ad) intestine tumors/mouse Avg. large  2.96 ±0.2^(b)  2.75 ± 0.46^(ab)  3.03 ± 0.52^(a)  3.01 ± 0.20^(a)  2.48 ±0.39^(ab)  2.57 ± 0.30^(ab)  1.50 ± 0.00^(b) 2.00 ± 0.00^(ab) intestinetumor size Avg. large  3.70 ± 1.51^(ab)  2.15 ± 0.83^(bcd)  3.47 ±1.29^(ab)  5.06 ± 0.85^(a)  2.25 ± 0.75^(bcd)  3.25 ± 1.34^(abc)  0.30 ±0.20^(d) 0.44 ± 0.29^(cd) intestine tumor load Total small   337   364  409   280   176   421   185   29 intestine tumors/group Avg. small 33.7 ± 4.5^(ab)  36.4 ± 6.3^(ab)  45.4 ± 8.2^(a)  28.0 ± 5.6^(bc)  17.6± 2.2^(cd)  46.8 ± 7.1^(a)  18.5 ± 1.9^(c)  3.2 ± 1.1^(d) intestinetumors/mouse Avg. small  1.32 ± 0.05^(a)  1.23 ± 0.04^(ab)  1.24 ±0.03^(ab)  1.16 ± 0.04^(ab)  1.05 ± 0.03^(b)  1.30 ± 0.04^(ab)  1.07 ±0.02^(ab) 1.25 ± 0.29^(ab) intestine tu- mor size (mm) Avg. small 43.62± 5.3^(ab) 45.67 ± 8.66^(ab) 57.84 ± 11.70^(a) 33.15 ± 8.09^(bc) 18.34 ±2.04^(cd) 61.62 ± 10.36^(a) 19.90 ± 2.11^(cd) 3.64 ± 1.12^(d) intestinetumor load Total Avg.  35.0 ± 4.5^(ab)  37.2 ± 6.6^(ab)  46.6 ± 8.3^(a) 29.8 ± 5.9^(bc)  18.5 ± 2.2^(c)  48.0 ± 7.4^(a)  18.7 ± 1.9^(c)  3.4 ±1.2^(d) tumors/mouse Total Avg.  1.32 ± 0.04^(a)  1.21 ± 0.03^(bd)  1.28± 0.04^(ab)  1.20 ± 0.04^(bc)  1.11 ± 0.04^(cd)  1.33 ± 0.04^(a)  1.08 ±0.02^(d) 1.04 ± 0.08^(d) overall tumor size (mm) Total Avg. 45.91 ±5.74^(ab) 45.97 ± 8.88^(ab) 61.30 ± 12.41^(a) 35.60 ± 7.10^(bc) 20.28 ±2.23^(cd) 64.84 ± 11.05^(a) 20.19 ± 2.24^(cd) 3.64 ± 1.27^(d) overalltumor load

[0062] As shown in Table 6, the analyses demonstrated that SDA waseffective in decreasing polyp number (47%), polyp size (18%) and polypload (number×size) (56%) in the large intestine and small intestine. Itshould be noted that while the terms polyp and tumor are usedinterchangeably, technically speaking, the lesions are polyps (i.e.,early stage tumors or neoplasms).

[0063] Unexpectedly, the effectiveness of SDA in inhibiting polypformation and development in the large intestine was comparable to thatof sulindac, a NSAID commonly used as a positive control, and wasgreater than that of not only ALA, but also EPA and DHA. ALA and EPAwere marginally efficacious, while DHA showed no efficacy in the largeintestine. Likewise, unexpectedly the effectiveness of SDA in inhibitingpolyp formation and development in the small intestine was comparable tothat of EPA, and was greater than that of ALA and DHA. This is alsoshown in bar-graph form in FIG. 10. GLA and CLA, in contrast to SDA, EPAand DHA, appeared to increase polyp number; however, the differenceswere not significant relative to the US17 control.

EXAMPLE 9 Effect of n-3 and n-6 PUFAs on Tissue Levels of ArachidonicAcid in the Small Intestine

[0064] The efficacy of select n-3 and n-6 polyunsaturated fatty acids inreducing the level of arachidonic acid in small intestine tissue wasevaluated using the Min/+mouse model. The following fatty acid ethylesters were tested for their effect on intestinal fatty acidcomposition: 1) ALA-EE; 2) SDA-EE; 3) EPA-EE; 4) DHA-EE; 5) GLA-EE; and6) CLA-EE. Fatty acid composition was determined in the small intestinebecause that is where the vast majority of polyps form.

[0065] These fatty acid esters were added to the US17 diet as discussedin Example 8. The arachidonic acid level in the phospholipid fraction ofthe small intestines of the mice was determined by gas chromatography.The results of this analysis are presented in FIG. 11. In FIG. 11, barslabeled with the same letter (e.g., control and CLA, both labeled with a“b”) have values which, statistically, are not different.

[0066]FIG. 11 shows that SDA was more effective than ALA, EPA and DHA indecreasing the level of arachidonic acid in the small intestine of themice. Decreasing the level of arachidonic acid in tissues is desirablebecause arachidonic acid metabolites have been implicated intumorigenesis (e.g., prostaglandins, leukotrienes, and HETEs).

EXAMPLE 10 Effect of Stearidonic Acid on Primary Tumor Growth in theNude Mouse/HT-29 Cancer Model

[0067] The efficacy of stearidonic acid (18:4n-3) in inhibiting primarytumor growth was evaluated using the nude mouse/HT-29 model. The nudemouse/HT-29 model has been described previously. Hernandez-Alcoceloa R.,Fernandez, F., Lacal, J C, Cancer Res., 59(13), 3112-18 (1999); Fantini,J., Cancer J., 5(2) (1992).

[0068] Nude (i.e., immunodeficient) mice were fed the US17 diet forthree weeks. HT-29 cells were cultured in RPMI-1640 medium supplementedwith fetal bovine serum, penicillin, and streptomycin (Gibco, GrandIsland, N.Y.) and maintained in a CO₂ atmosphere at 37° C. Afterachieving the optimal cell density, the HT-29 cells were rinsed and thensuspended in phosphate buffered saline (PBS). A cell suspension was madein MATRIGEL (Becton Dickinson Labware, Bedford, Mass.). The suspensionwas ⅔ by volume cells in PBS and ⅓ by volume MATRIGEL. MATRIGEL providesan extracellular matrix secreted by endothelial cells. The matrixcontains angiogenic and cell proliferation growth factors that aid inHT-29 cell attachment and proliferation as a primary tumor.

[0069] One million cells were injected in a 30 μl volume into thesubplanter area of the righthind footpad of the nude mice. Five daysafter the HT-29 cell injections, half of the mice were switched to aUS17 diet containing stearidonic acid (3% wt.=10 g/day human equivalentdose) in place of oleic acid. The amount of primary tumor growth wasmeasured by measuring the change in mouse footpad volume over time.Footpad volume was measured with a plethysmometer (Ugo Basile,Camerio-Varese, Italy). The results of these measurements are set forthin FIG. 12.

[0070]FIG. 12 shows that the mice fed a US17 diet containing stearidonicacid exhibited decreased primary tumor growth as compared to those fedthe US17 control diet. After 35 days, the mice fed the US17 dietcontaining SDA exhibited 33% less primary tumor growth than those fedthe US17 diet.

[0071] Other variations and modifications of this invention will beobvious to those skilled in the art. This invention is not limited,except as set forth in the claims.

What is claimed is:
 1. A method for treating or preventing cancer in amammal, said method comprising administering to a mammal in need thereofa cancer inhibiting amount of a metabolite of α-linolenic acid selectedfrom the group consisting of stearidonic acid (18:4 n-3),eicosatetraenoic acid (20:4 n-3), docosapentaenoic acid (22:5 n-3) andmixtures thereof.
 2. A method according to claim 1, wherein said canceris colon cancer, breast cancer, prostate cancer or lung cancer.
 3. Amethod according to claim 1, wherein said metabolite comprisesstearidonic acid (18:4 n-3).
 4. A method according to claim 1, whereinsaid metabolite consists essentially of stearidonic acid (18:4 n-3). 5.A method according to any one of claims 1-4, wherein said metabolite isin the form of a lipid, a carboxylate salt, an ester, a triglyceride, anamide or another pharmacologically acceptable carboxylic acidderivative.
 6. A method according to any one of claims 1-4, wherein saidmetabolite is in the form of a triglyceride.
 7. A method according toany one of claims 1-4, wherein said metabolite is in the form of anester.
 8. A method according to any one of claims 1-4, wherein saidmetabolite is administered in a dosage amount from about 1 mg/Kg/day toabout 300 mg/Kg/day.
 9. A method according to any one of claim 5,wherein said metabolite is administered in a dosage amount from about 1mg/Kg/day to about 300 mg/Kg/day.
 10. A method according to any one ofclaim 6, wherein said metabolite is administered in a dosage amount fromabout 1 mg/Kg/day to about 300 mg/Kg/day.
 11. A method according toclaim 5, wherein said metabolite is administered in a dosage amount fromabout 10 mg/Kg/day to about 150 mg/Kg/day.
 12. A method according toclaim 6, wherein said metabolite is administered in a dosage amount fromabout 10 mg/Kg/day to about 150 mg/Kg/day.
 13. A method according toclaim 7, wherein said metabolite is administered in a dosage amount fromabout 10 mg/Kg/day to about 150 mg/Kg/day.
 14. A method according toclaim 8, wherein said metabolite is administered in a dosage amount fromabout 10 mg/Kg/day to about 150 mg/Kg/day.
 15. A method according to anyone of claims 1-4, wherein said metabolite is administered in apharmaceutical preparation, a nutritional preparation or a foodpreparation.
 16. A method according to claim 15, wherein said metaboliteis administered in a food preparation.